Apolipoprotein E (apoE) is is highly inhibitory toward atherosclerosis, but the mechanisms of this inhibition remain poorly understood. In the first cycle of this P01, we showed that human apoE3 inhibited S-phase entry of primary murine vascular smooth muscle cells (VSMCs) through upregulation of COX-2 and promotion of prostacyclin production with ligation of the prostacyclin receptor (IP). The experiments in this new project in this P01 will continue to explore the mechanisms by which apoE signals to upregulate COX-2 and ultimately to inhibit VSMC proliferation, and we will explore the in vivo consequences of this novel effect of apoE. Specific Aim 1: To determine the structure-function properties of apoE necessary and sufficient to confer the ability to upregulate COX-2 and inhibit VSMC proliferation. We will systematically assess the effect of lipid association with apoE on its ability to upregulate COX-2. We will localize the domain(s) within apoE necessary and sufficient for upregulation of COX-2 and inhibition of VSMC proliferation. We will compare the three major human apoE isoforms with regard to their ability to upregulate COX-2 and to inhibit proliferation. We will perform selected site-directed mutagenesis to determine the importance of selected apoE properties, such as formation of the four-helix binding, lipid binding, receptor binding, and heparin binding to the ability to upregulate COX-2 and inhibit proliferation. Specific Aim 2: To determine the cell surface receptor(s) that are responsible for mediating apoE's effects on COX-2 upregulation and VSMC proliferation. We will systematically test, through the use of VSMCs from gene targeted mouse models and through antibody inhibition, the dependence of the ability of apoE to induce COX-2 expression on known apoE receptors such as the LDL receptor, LRP, the VLDL receptor, and the apoE2 receptor, as well as different HSPGs. We will also test the hypothesis that apoE is less effective in inhibiting VSMC proliferation in vivo in response to vascular injury as well as atherosclerosis itself in mice lacking these receptor(s). Specific Aim 3: To determine the molecular mechanisms by which apo E upregulates COX-2 expression in vascular smooth muscle cells. We will characterize the kinetics of effect of apoE in regulating COX-2 expression. We will establish the dependence of the apoE effect on COX-2 on NF-kappaB and cAMP-dependent pathways. We will seek other signaling pathways that are responsible for the apoE effect on COX-2 regulation. We will use proteomic and genomic approaches to investigate in an unbiased fashion the effects of apoE on VSMCs. The proposed experiments will advance our understanding of the mechanisms by which apoE upregulates COX-2 and inhibits vascular smooth muscle cell proliferation. These mechanisms could be important for the ability to apoE to inhibit atherosclerosis and may provide novel insights into the molecular mechanisms by which apoE exerts its myriad of biological effects.